Genotyping is the mandatory starting point for most genetic analyses, both basic and applied, and has contributed decisively to the development of genetics and its applications. The genotyping of polymorphic DNA loci has recently been facilitated by two fundamental advances: the invention of the Polymerase Chain Reaction (PCR) (Saiki et al., 1985; Mullis et al., 1986) and the discovery of a fairly even spread of polymorphic DNA sequences throughout genomes (mainly DNA markers composed of tandem arrays of motifs one or more base pairs long) (Dib et al., 1996; Tóth et al., 2000). Both advances have contributed decisively towards understanding of the structure and function of genomes of living organisms, and towards the development of population genetics, genetic epidemiology, genome mapping, genetic therapy, disease diagnosis, preimplantational genetic diagnosis, forensic analysis, tissue and organ transplantation, pharmacogenetics, paternity testing, etc.
The conventional methods used for genotyping polymorphic DNA loci are based on the following strategy: (a) amplifying the locus alleles present in a sample (generally by means of PCR); (b) identifying the amplified alleles (generally by means of electrophoresis); and (c) determining the sample genotype from the amplified alleles.
This genotyping strategy should guarantee that the genotype determined from amplification products is in fact the actual sample genotype. In practice, however, conventional genotyping methods carry associated a percentage of error, which is dependent on the experimental conditions used.
Genotyping errors have very negative consequences in genetic analyses and in the applications derived from such analyses. Obviously, genotyping errors may have serious consequences if they lead to the erroneous identification of human individuals (e.g. in criminal analyses, in paternity testing, etc.). Likewise, genotyping errors may lead to wrong medical diagnoses which may be especially pernicious in disease diagnosis, preimplantational diagnosis, tissue and organ transplantation, etc. On the other hand, it is known that genotyping errors negatively influence the accuracy of genetic analyses, leading to serious drawbacks such as loss of power in gene-mapping studies, significant distortion of map distances and false exclusion of the true location of a disease-predisposing gene (Buetow, 1991; Goldstein et al., 1997; Göring and Terwilliger, 2000). The negative effects of genotyping errors may be even greater in gametic disequilibrium analyses (Zapata et al., 2001a; Zapata et al., 2001b), because a single genotyping error can destroy evidence of past non-recombinant meioses (Terwilliger et al., 1997; de La Chapelle and Wright, 1998; Göring et al., 1997; Göring and Terwilliger, 2000; Akey et al., 2001).
An important source of genotyping errors is the occurrence of problems during amplification that lead to the total or partial absence of the amplification product of, at least, one of the alleles originally present in the sample. The partial absence of the amplification product of an allele, or partial non-amplification, is also known as preferential amplification (Walsh et al., 1992). The total absence of the amplification product of an allele, or total non-amplification, is known as allele dropout (Findlay et al., 1995; Gagneux et al., 1997) or null allele (Pemberton et al., 1995).
The occurrence of this type of problem during the amplification procedure has serious consequences. In the case of heterozygous diploid samples, total non- amplifications and some partial non-amplifications lead to the incorrect genotyping of the sample as homozygous (Demers et al., 1995; Findlay et al., 1995; Pemberton et al., 1995; Fishback et al., 1999; Anderson et al., 2000).
In view of the serious consequences of genotyping errors in genetic analyses and in genetic applications, considerable efforts have been made:
1.—to identify the causes of total and partial non-amplifications;
2.—to detect and/or prevent genotyping errors due to total and partial non-amplifications.
1.Causes of total and partial non-Amplifications
Recent analyses have revealed the common causes of total and partial non-amplification. The partial non-amplification of one allele in diploid samples can result from several factors such as: (a) significant GC% differences between alleles from a heterozygous sample (these differences can allow the denaturation of one allele but not the other, phenomenon known as differential denaturation) (Walsh et al., 1992); (b) between-allele length differences, resulting in the preferential amplification of the shorter allelic product (probably favoured when Taq polymerase is limiting) (Walsh et al., 1992); (c) stochastic fluctuations in the number of copies of each allele (Walsh et al., 1992); (d) mismatches between the primers and one allele (which may result from mutations in the priming region) (Walsh et al., 1992); (e) damage to the DNA template (by ultraviolet irradiation or by monovalent salts such as sodium chloride, sodium acetate or ammonium acetate) (Mutter and Boynton, 1995); (f) low-stringency primer annealing (which may result from the coamplification either of the locus and an internal control for sizing the amplified alleles, or of more than one polymorphic sequence) (Weissensteiner and Lanchbury, 1996).
It has been suggested that total non-amplification may result from extreme partial non-amplification (Findlay et al., 1995; Ronai et al., 2000). Therefore, mechanisms that lead to partial non-amplification can also lead to total non-amplification. In addition, other evidence shows that the total non-amplification rate is especially high when template concentration in the sample is low (Gagneux et al., 1997; Lissens and Sermon, 1997). This is the case when the genotyping is done either from a single cell (for instance, in preimplantational genetic diagnosis), or from biological remains that are degraded and/or retain little DNA (as may occur in several forensic analyses). In these cases, total non-amplification is favoured by factors such as (a) chromosomal mosaicism, and more specifically the presence of haploid cells in an otherwise diploid embryo (Harper et al., 1995); (b) the method used for lysing the cell before PCR (Lissens and Sermon, 1997, and references therein); (c) the denaturation temperature at the start of the first PCR cycle (Ray and Handyside, 1996); (d) DNA breaks produced by endogenous endonucleases (Lissens and Sermon, 1997); (e) suboptimal PCR conditions (Handyside and Delhanty, 1997); and (f) rapid degradation of the target DNA during thermocycling (Handyside and Delhanty, 1997).
2. Detection and/or Prevention of Genotyping Errors Due to Total or Partial Non-Amplifications
Several improvements of conventional genotyping methods have been proposed for detecting and/or preventing genotyping errors due to total and partial non-amplifications. Basically, these may be divided into two categories: (2.1) direct methods and (2.2) indirect methods.
2.1. Direct Methods
Direct methods are based on (a) improvement of amplification conditions for preventing the occurrence of total or partial non-amplifications and (b) improvement of the system for the detection of amplified alleles.
(a) Improvement of amplification conditions
Several optimizations of amplification conditions have been suggested for trying to prevent the occurrence of partial non-amplifications, including: (a) not using either a DNA template concentration higher than 200 ng or an extension time longer than 2 min (Deka et al., 1992); (b) substitution of dGTP either for 7-deaza-2′-dGTP (Mutter and Boynton, 1995) or for 2′-desoxyinosine-5′-triphosphate (dITP) (Ronai et al., 2000); (c) using PNA (Peptide Nucleic Acid) during the amplification procedure (Demers et al., 1995); (d) using double-strand-destabilizing cosolutes such as betaine and glycerol (Weissensteiner and Lanchbury, 1996); (e) increasing the KCl concentration (Fishback et al., 1999).
Optimizations of amplification conditions that have been suggested for reducing the occurrence of partial non-amplifications include: (a) increasing the denaturation temperature (Lissens and Sermon, 1997); (b) carrying out whole-genome amplification by primer extension pre-amplification (PEP) followed by nested PCR (Handyside and Delhanty, 1997); (c) approaches for increasing the DNA-template amount, such as biopsy of more than one embryo cell (Harper et al., 1996), or increase in the number of fetal cells isolated from maternal fluids (Garvin et al., 1998), etc.
(b) Improvement of the System for the Detection of Amplified Alleles
Similarly, improvements in the system for the detection of amplified alleles have been suggested for the detection of genotyping errors due to total and partial non-amplifications. For instance, Findlay et al. (1995) have demonstrated that fluorescent PCR decreases the genotyping error rate due to total and partial non-amplifications. This is because fluorescent PCR, which is much more sensitive than conventional PCR, allows detection of samples which are classified by conventional PCR as homozygotes but which are in fact heterozygous with extreme partial non-amplification.
In this connection, it has been pointed out that the definitive method for high-resolution genotyping is the detection of polymorphisms through fluorescent-automatic-sequencing by the use of automated sequencers based on capillary electrophoresis, such as the ABI PRISM 310 or the ABI PRISM 3100 (Applied Biosystems, 2001). This approach could potentially detect genotyping errors due to partial non-amplifications. Nevertheless, the genotyping problem associated with total non-amplification remains unresolved when automated sequencers such as the ABI PRISM 310 are used (Garvin et al., 1998). In fact, several authors have recently indicated that genotyping errors are inevitable (Douglas et al., 2000; Göring and Terwilliger, 2000) and more difficult or impossible to detect when Mendelian information is unavailable (Weeks et al., 2000).
Consequently, the suggested improvements of amplification conditions or of the detection system do not eliminate all partial or total non-amplifications, and do not ensure detection of all genotyping errors due to these PCR errors.
2.2. Indirect methods
An alternative for detecting genotyping errors due to total or partial non-amplification is to resort to indirect methods such as: (a) replicating the amplification a sufficient number of times to obtain a reliable genotype, and (b) Mendelian analysis.
(a) Amplification replicates
It has been suggested that three replicates of one sample (i.e., repeating the amplification and detection of amplified alleles three times) should detect 95% of genotyping errors due to total or partial non-amplifications (Gagneux et al., 1997). Taberlet et al. (1996) have suggested that this percentage may be increased to 99% when seven replicates are performed, although this remains controversial (Weissensteiner, 1997; Taberlet, 1997).
Nevertheless, this strategy has two main limitations. First, it does not allow a priori identification of the samples with total or partial non-amplification. Therefore, it is necessary to carry out several PCR replicates of all apparent homozygotes, given that conventional genotyping methods do not distinguish between homozygous samples and heterozygous samples with total non-amplification. Obviously, this approach is hardly applicable in studies in which a large number of individuals are analyzed (e.g. in genome scans and in many population and epidemiological analyses). Second, the applicability of this strategy is seriously limited by DNA availability, because the amount of DNA is often very reduced (for instance, in preimplantational genetic analyses, in forensic studies in which samples are degraded or contain very little DNA, as in the case of hairs, etc.).
(b) Mendelian analysis
Another indirect method valuable for detecting a broad range of genotyping errors is Mendelian analysis (Ewen et al., 2000). However, this procedure is not always applicable, because samples biologically related with the sample of interest are not always available. Moreover, genotyping errors may not always be readily detectable as inconsistencies in Mendelian inheritance, especially in studies of small families (Weeks et al., 2000).
In view of the above, it is evident that conventional methods, based on genotyping from amplified alleles, are not effective for detecting and/or preventing genotyping errors due to total or partial non-amplification. Notably, such errors occur even after the use of direct and indirect methods for detecting and/or preventing them specifically. There is thus a clear need for new genotyping methods that are effective for detecting and/or preventing genotyping errors due to total or partial non-amplifications.
Recent evidence suggests that several products generated by PCR during allele amplification (particularly heteroduplexes) may be used as an additional source of information in the identification of heterozygous samples (Wilkin et al., 1993; Neilan et al., 1994; Ardren et al., 1999) and in the detection of homoplasy (Szibor et al., 1996; Haddad et al., 1997; Ardren et al., 1999). As far as we know, however, these reports have not considered the possibility that additional products other than the amplified alleles themselves may be of value in the detection and/or prevention of genotyping errors originating from total or partial non-amplifications.